Project Summary Influenza viruses constantly undergo antigenic changes which pose great challenges for development of vaccines against influenza epidemics as well as pandemics. There are at least 16 known subtypes of influenza A viruses which fall into two broad phylogenetic groups. Current trivalent inactivated vaccines (TIV; H1, H3, and B) for seasonal influenza are not suited for controlling pandemics as they are strain-specific and have to undergo annual strain exchange in order to match circulating strains. A broad-spectrum influenza vaccine which is at least effective against major subtypes important to epidemics and having a greater potential to cause future pandemics is greatly needed to meet the current and immediate needs. KJ Biosciences proposes a novel and highly practical solution to meet this urgent unmet need - a broad-spectrum influenza vaccine based on inactivated antigens treated at proper low pH conditions. Our strategy is based on the novel finding that inactivated antigens treated at appropriate low pH conditions can induce increased cross-reactive antibody responses and cross protection against heterologous viruses of the same or different subtypes. In particular, we have found that it is the inactivated antigens treated at the mild low pH conditions (low pH at low temperatures) with a partial potency loss that can uniquely induce the greater cross reaction with HA2, the more conserved part of HA, and cross protection. Such antigens can therefore be used for a novel broad-spectrum influenza vaccine. The new vaccine can be readily produced to meet strain- specific potency standard so that it will not only provide the same level of the strain-specific protection against viruses contained in the vaccine as the current inactivated vaccines, but also cross protection against viruses not contained in the vaccine, including possible pandemic as well as seasonal variant viruses. It is particularly well suited for TIV since an even broader cross-reactivity could be obtained by using treated antigens for all three strains, especially when considering that H1 and H3 subtypes belong separately to the two different phylogenetic groups. Importantly, the new TIV can be readily incorporated into the current immunization programs against seasonal influenza. The effectiveness and manufacturing processes for current inactivated vaccines are well established. Thus, the new inactivated vaccines based on antigens treated at proper low pH conditions could be developed much faster, providing a timely and effective counter measure for possible pandemics as well as better control of seasonal influenza. In addition, the low pH treatment at proper conditions can also be potentially used to convert existing vaccines in circulation or stockpiles just prior to administration to provide cross protection in emergencies such as a pandemic outbreak caused by a newly emerged variant virus. In light of these promising novel findings and significant potential benefits, KJ Biosciences propose to continue developing this new inactivated influenza vaccine based on antigens treated at proper low pH conditions. Our proposed studies include two specific aims: Specific Aim 1 - Characterization and optimization of low pH-treated antigens, and Specific Aim 2 - Immunogenicity and cross- protective effect of low pH-treated antigens to demonstrate the increased cross-reactive immune responses and cross protection with antigens treated at the optimal low pH conditions. Successful completion of these two specific aims will form the foundation for future development activities including the pre-IND meeting with the FDA to lay out the development path for this new vaccine and subsequent preclinical and clinical safety and immunogenicity studies.